Risk assessment and predictors of the exposure to polycyclic aromatic hydrocarbons in Spanish adults by urinary human biomonitoring.

Polycyclic aromatic hydrocarbons (PAHs) are produced primarily during incomplete combustion of organic matter and in various industrial processes. They are widespread environmental pollutants that are of significant interest due to their potential toxicity. Humans can be exposed to PAHs through ingestion, inhalation and dermal contact. In the present study, ten urinary PAH biomarkers were determined in first-morning urine samples (n = 504) from the adult population (aged 18-65 years) residing in the Valencian Region of Spain. These samples were analysed using liquid-liquid extraction followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). All PAH biomarkers were quantified in more than 65 % of the urine samples. Naphthalene biomarkers, 1-hydroxynaphthalene (1OHNAP) and 2-hydroxynaphthalene (2OHNAP), exhibited the highest levels with geometric means (GMs) of 0.7 and 11.9 µg L-1, respectively. The 95th percentile of all PAH biomarkers ranged from 0.22 to 64.8 µg L-1. Estimated daily intakes (EDIs) for the analysed PAH families in the studied population ranged from 17 (pyrene) to 18581 (naphthalene) ng·kg-bw-1·day-1 (GM). Significant associations were observed between the quantified urinary metabolites of PAHs and smoking status, home location, annual household incomes, BMI, and the consumption of grilled food in the last 24 h. Hazard quotients (HQs) of naphthalene and consequently, hazard indexes (HIs) were above 1, especially for smokers. Therefore, potential health risks associated with PAH exposure in the target population could not be discarded.

Assessment of acrylamide exposure in Spain by human biomonitoring: Risk and predictors of exposure.

Acrylamide (AA), a chemical compound currently classified as "reasonably anticipated to be a human carcinogen", is formed through the Maillard reaction in processed carbohydrate-rich foods and is also present in tobacco smoke. The primary sources of AA exposure in the general population are dietary intake and inhalation. Within a 24-h period, humans eliminate approximately 50% of AA in the urine, predominantly in the form of mercapturic acid conjugates such as N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2- hydroxyethyl)-L-cysteine (GAMA3), and N-acetyl-3-[(3-amino-3-oxopropyl)sulfinyl]-L-alanine (AAMA-Sul). These metabolites serve as short-term biomarkers for AA exposure in human biomonitoring studies. In this study, we analysed first-morning urine samples from the adult population (aged 18-65 years) residing in the Valencian Region, Spain, (n = 505). AAMA, GAMA-3 and AAMA-Sul were quantified in 100% of the analysed samples, with geometric means (GM) of 84, 11 and 26 µg L-1, respectively, while the estimated daily intake of AA in the studied population ranged from 1.33 to 2.13 µg·kg-bw-1·day-1 (GM). Statistical analysis of the data indicated that the most significant predictors of AA exposure were smoking and the amount of potato fried products and, biscuits and pastries consumed last 24 h. Based on risk assessment approaches conducted, the findings suggest that exposure to AA could pose a potential health risk. Therefore, it is crucial to closely monitor and continuously evaluate AA exposure to ensure the well-being of the population.

Assessment of exposure to pesticide mixtures in five European countries by a harmonized urinary suspect screening approach.

Humans are exposed to a mixture of pesticides through diet as well as through the environment. We conducted a suspect-screening based study to describe the probability of (concomitant) exposure to a set of pesticide profiles in five European countries (Latvia, Hungary, Czech Republic, Spain and the Netherlands). We explored whether living in an agricultural area (compared to living in a peri-urban area), being a a child (compared to being an adult), and the season in which the urine sample was collected had an impact on the probability of detection of pesticides (-metabolites). In total 2088 urine samples were collected from 1050 participants (525 parent-child pairs) and analyzed through harmonized suspect screening by five different laboratories. Fourty pesticide biomarkers (either pesticide metabolites or the parent pesticides as such) relating to 29 pesticides were identified at high levels of confidence in samples across all study sites. Most frequently detected were biomarkers related to the parent pesticides acetamiprid and chlorpropham. Other biomarkers with high detection rates in at least four countries related to the parent pesticides boscalid, fludioxonil, pirimiphos-methyl, pyrimethanil, clothianidin, fluazifop and propamocarb. In 84% of the samples at least two different pesticides were detected. The median number of detected pesticides in the urine samples was 3, and the maximum was 13 pesticides detected in a single sample. The most frequently co-occurring substances were acetamiprid with chlorpropham (in 62 urine samples), and acetamiprid with tebuconazole (30 samples). Some variation in the probability of detection of pesticides (-metabolites) was observed with living in an agricultural area or season of urine sampling, though no consistent patterns were observed. We did observe differences in the probability of detection of a pesticide (metabolite) among children compared to adults, suggesting a different exposure and/or elimination patterns between adults and children. This survey demonstrates the feasibility of conducting a harmonized pan-European sample collection, combined with suspect screening to provide insight in the presence of exposure to pesticide mixtures in the European population, including agricultural areas. Future improvements could come from improved (harmonized) quantification of pesticide levels.

Comprehensive suspect screening for the identification of contaminants of emerging concern in urine of Flemish adolescents by liquid chromatography high-resolution mass spectrometry.

The increasing human exposure to contaminants of emerging concern (CECs) cannot be fully assessed by targeted biomonitoring methods alone as these are limited to a subset of known analytes. On the contrary, suspect screening approaches based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) allow the simultaneous detection of a high number of CECs and/or their (predicted) metabolites leading to a more comprehensive assessment of possible human exposure to these compounds. Within this study, 83 urine samples of Flemish adolescents (47 males, 36 females) collected in the frame of the 4th cycle of the Flemish Environment and Health Study (FLEHS IV) were selected with the aim of including a high and a low exposure group based on the overall exposure of 45 known contaminants. Samples were analyzed using a previously developed method involving a suspect screening approach to annotate CECs and their metabolites. The applied suspect list contained a total of >12,500 CECs and their known and predicted metabolites resulting from metabolization reactions, such as hydroxylation, glucuronidation and methylation. In total, 63 compounds were annotated at a confidence level of 3 or better, with most of the detected compounds not included in current biomonitoring programs. 5 out of the 63 compounds could be assigned with confidence level 2. Five compounds could unequivocally be identified (confidence level 1) through the comparison with reference standards. Personal care products were the main detected compound class (42% of detected compounds). Additionally, a detailed literature search indicated potential toxic effects for several of the detected CECs. Lastly, in the urine samples, a significantly higher number (p < 0.05) of compounds was detected in the high exposure group as opposed to the low exposure group. This difference could only be observed between high and low exposure load samples of female participants (p < 0.01).

Comprehensive investigation of persistent and mobile chemicals and per- and polyfluoroalkyl substances in urine of flemish adolescents using a suspect screening approach.

Persistent and mobile chemicals (PMs) and per- and polyfluoroalkyl substances (PFAS) are groups of chemicals that have received recent global attention due to their potential health effects on the environment and humans. In this study, exposure to a broad range of PMs and PFAS was investigated in Flemish adolescents' urine samples (n = 83) using a suspect screening approach. For this purpose, three sample preparation methods were evaluated, and a basic liquid-liquid extraction was optimized for urine analysis based on the extraction efficiency of PMs (53-80%) and PFAS (>70%). In total, 9 PMs were identified in urine samples at confidence levels (CL) 1-3 and, among them, acetaminophen, 4-aminophenol, 2,2,6,6-tetramethyl-4-piperidone, trifluoroacetic acid (TFAA), sulisobenzone, ethyl sulfate, and 1,2-benzisothiazol-3(2H)-one 1,1-dioxide were confirmed at CL 1 and 2. In addition, the detection and identification of 2,2,6,6-tetramethyl-4-piperidone, 4-aminophenol, TFAA, and m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl) aniline (CL 3), has been reported for the first time in human urine in this study. For PFAS, only 2 compounds were identified at CL 4, implying that urine is not a suitable matrix for suspect screening of such compounds. A significant difference between sexes was observed in the detection rate of identified PMs, in particular for acetaminophen, 4-aminophenol, and sulisobenzone. The findings of this study can be used in future human biomonitoring programs, such as by including the newly identified compounds in quantitative methods or monitoring in other human matrices (e.g., serum).

A large scale multi-laboratory suspect screening of pesticide metabolites in human biomonitoring: From tentative annotations to verified occurrences.

Within the Human Biomonitoring for Europe initiative (HBM4EU), a study to determine new biomarkers of exposure to pesticides and to assess exposure patterns was conducted. Human urine samples (N = 2,088) were collected from five European regions in two different seasons. The objective of the study was to identify pesticides and their metabolites in collected urine samples with a harmonized suspect screening approach based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) applied in five laboratories. A combined data processing workflow included comprehensive data reduction, correction of mass error and retention time (RT) drifts, isotopic pattern analysis, adduct and elemental composition annotation, finalized by a mining of the elemental compositions for possible annotations of pesticide metabolites. The obtained tentative annotations (n = 498) were used for acquiring representative data-dependent tandem mass spectra (MS2) and verified by spectral comparison to reference spectra generated from commercially available reference standards or produced through human liver S9 in vitro incubation experiments. 14 parent pesticides and 71 metabolites (including 16 glucuronide and 11 sulfate conjugates) were detected. Collectively these related to 46 unique pesticides. For the remaining tentative annotations either (i) no data-dependent MS2 spectra could be acquired, (ii) the spectral purity was too low for sufficient matching, or (iii) RTs indicated a wrong annotation, leaving potential for more pesticides and/or their metabolites being confirmed in further studies. Thus, the reported results are reflecting only a part of the possible pesticide exposure.

Risk assessment of the exposure of Spanish children to acrylamide using human biomonitoring.

Acrylamide (AA) is an organic contaminant that naturally forms in starchy foods during high-temperature cooking under low-moisture conditions. It is mainly produced from the sugars and amino acids present in food by the Maillard reaction. When humans are exposed to AA, AA is eliminated in the urine as mercapturic acid conjugates, primarily including N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA3), and N-acetyl-3-[(3-amino-3-oxopropyl)sulfinyl]-L-alanine (AAMA-Sul), which are used as exposure biomarkers of AA in human biomonitoring studies. Although the carcinogenic effects of AA on humans have not been demonstrated yet, some studies have shown that AA may negatively affect children's health. The main objective of this study was to evaluate the exposure of Spanish children (n = 612) to AA. For this purpose, the levels of AAMA, AAMA-Sul, and GAMA3 in first-morning urine samples were analyzed by "dilute and shoot" and liquid chromatography coupled to tandem mass spectrometry. The three metabolites were detected in all the children involved in this study in the following order (geometric mean (GM)): AAMA (79 ng ml-1) > AAMA-Sul (28 ng ml-1) > GAMA3 (18 ng ml-1). Statistical analysis suggested that the intake of fried potato products and biscuits could be associated with higher levels of AA metabolites in urine. Estimated daily intakes of AA in the children under study were in the range of 1.2-1.5 µg AA·kg-body weight-1·day-1 (GM). Risk assessment calculations indicate that the health risk of AA exposure cannot be overlooked and the exposure of Spanish children to AA should be closely monitored.

Review of the state of the art of acrylamide human biomonitoring.

Human biomonitoring (HBM) is a very useful tool for assessing human exposure to acrylamide (AA). In the framework of the Human Biomonitoring Initiative (HBM4EU) AA was included in its second list of priority substances due to the potential threat to human health. HBM data on AA are scarce, but the use of specific and sensitive biomarkers represents a reliable indicator of exposure. In this review an overview of available knowledge on HBM of AA is provided in terms of: i) preferred exposure biomarkers and matrices for the HBM of AA; ii) analytical methods for determining its biomarkers of exposure in the most used specimens; iii) current HBM data available; and iv) tools for interpreting HBM data for AA in relation to risk assessment. Finally, future trends in this field are discussed.

Health Risk Assessment of Exposure to 15 Essential and Toxic Elements in Spanish Women of Reproductive Age: A Case Study.

This case study investigates the exposure of 119 Spanish women of reproductive age to 5 essential (Co, Cu, Mn, V, Zn) and 10 toxic (Ba, Be, Cs, Ni, Pb, Pt, Sb, Th, Al, U) elements and assesses their risk. The essential elements (Co, Cu, Mn, V, and Zn) showed average concentrations (GM: geometric mean) of 0.8, 35, 0.5, 0.2, and 347 µg/L, respectively. Five of the toxic elements (Ba, Cs, Ni, Al, U) exhibited detection frequencies of 100%. The GM concentrations of the novel toxic elements were 12 µg/L (Al), 0.01 µg/L (Pt), 0.02 µg/L (U), 0.12 µg/L (Th), 0.009 µg/L (Be) and 4 µg/L (Cs). The urine analysis was combined with a survey to assess any variations between subgroups and potential predictors of exposure to elements in the female population. Significant differences were obtained between the rural and urban areas studied for the toxic element Cs, with higher levels found in mothers living in urban areas. In relation to diet, statistically significantly higher levels of essential (Cu) and toxic (Ba) elements were detected in women with a high consumption of fish, while mothers who consumed a large quantity of legumes presented higher levels of the toxic element Ni (p = 0.0134). In a risk-assessment context, hazard quotients (HQs) greater than 1 were only observed for the essential elements Zn and Cu in P95. No deficiency was found regarding the only essential element for which a biomonitoring equivalent for nutritional deficit is available (Zn). For the less-studied toxic elements (Al, Pt, U, Th, Be, and Cs), HQs were lower than 1, and thus, the health risk due to exposure to these elements is expected to be low for the female population under study.

Biomonitoring of Phthalates, Bisphenols and Parabens in Children: Exposure, Predictors and Risk Assessment.

Exposure to emerging contaminants, such as phthalates, bisphenols and parabens in children has been associated with possible neurodevelopment and endocrine alterations. In the present study, the biomonitoring of biomarkers in children (5-12 years old) from the Valencia Region (Spain) have been implemented using urines from the BIOVAL program. More than 75% of the children studied (n = 562) were internally exposed (>LOQ) to bisphenols and parabens, and the whole population assessed (n = 557) were exposed to at least one phthalate. The geometric means (GM) of the concentrations of bisphenol A, methyl paraben and propyl paraben were 0.9, 1.4 and 0.39 ng/mL, respectively. Regarding phthalates, monoethyl phthalate GM was 55.0 ng/mL and diethyl hexyl phthalate (as the sum of five metabolites) GM was 60.6 ng/mL. Despite the studied population being widely exposed, the detection frequencies and concentrations were in general lower than in previous studies involving children in Spain and in other countries in recent years. Furthermore, the risk assessment study concluded that the internal exposure to phthalates, bisphenols and parabens is lower than the guidance values established, and, therefore, a health risk derived from the exposure to these compounds in the studied population is not expected.

Biomonitoring of glyphosate and AMPA in the urine of Spanish lactating mothers.

The objectives of this study were to evaluate the urinary levels of Glyphosate (Gly) and its metabolite aminomethylphosphonic acid (AMPA) in Spanish breastfeeding mothers (n = 97), to identify the main predictors of exposure and to perform a risk assessment. Urine samples were analyzed using a method based on solid phase extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The developed method showed limits of quantification of 0.1 µg/L for both analytes. The detection frequencies (DFs) were 54% for Gly and 60% for AMPA, with geometric means (GMs) of 0.12 µg/L and 0.14 µg/L, respectively. In the statistical analysis, no relationship was found between the urinary levels of Gly and AMPA. Multiple regression analysis showed a significant relationship between the intake of eggs and fruits and Gly levels in urine. Estimated daily intakes (EDIs), hazard quotients (HQs), and a hazard index (HI) were calculated to analyze the obtained data from a health risk perspective. The GMs of the EDIs were 0.31 and 0.37 µg/kg of body weight (BW)/day for Gly and AMPA, respectively. The HQs were calculated considering 0.5 mg/kg BW/day as an acceptable daily intake (ADI), which EFSA has established as a health-based reference value for both analytes. The values obtained were lower than 1, and thus, low health risk due to Gly and AMPA exposure was expected for the population under study.

Children's exposure to polycyclic aromatic hydrocarbons in the Valencian Region (Spain): Urinary levels, predictors of exposure and risk assessment.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that are released into the environment during incomplete combustion of organic matter and which can have a negative effect on human health. PAHs enter the human body mostly through ingestion of food or inhalation of tobacco smoke. The purpose of the present study is to evaluate the internal levels of PAHs that children living in the Valencian Region (Spain) are exposed to. In total, we measured eleven biomarkers of exposure to naphthalene, fluorene, phenanthrene, pyrene, and benzo(a)pyrene in the urine of 566 children aged 5-12. The analytical method was based on a liquid-liquid extraction of the PAH metabolites from the urine samples, followed by their determination by liquid chromatography coupled to tandem mass spectrometry. In addition, we used a questionnaire to collect the socio-demographic characteristics and 72 h dietary recall information of the participants in our study. Overall, we detected PAH metabolites in more than 78% of the children, with the exception of 3-hydroxyfluorene and 3-hydroxybenzo(a)pyrene, which were found in less than 37% of the analyzed samples. The most abundant biomarker found was 2-hydroxynaphthalene, with a geometric mean of 10 ng·ml-1. Reference values (RV95) ranging from 0.11 (4-hydroxyphenanthrene) to 53 ng·ml-1 (2-hydroxynaphthalene) in urine of Spanish children were derived from the present study. According to the statistical analysis, the factors that were significantly associated with the internal exposure to PAHs were province of residence, body mass index (BMI), children's age, consumption of plastic-wrapped food, and dietary habits. The estimated daily intakes in geometric mean terms ranged from 5 (fluorene) to 204 ng·kg-bw-1·day-1 (naphthalene). Risk assessment calculations showed higher hazard quotients and hazard indexes for children aged 5-8 than those aged 9-12, but all were below 1. In conclusion, no potential non-cancer health risk due to PAH exposure was observed in children living in Spain.

Biomonitoring of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in human milk: Exposure and risk assessment for lactating mothers and breastfed children from Spain.

The main objective of the present research was to evaluate the levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in the human milk of Spanish lactating women who participated in the BETTERMILK project so that exposure and risk could be assessed for these mothers and their breastfed children. The total ∑PCDD/Fs + dl-PCBs TEQ2005 concentrations in the upper-bound (UB) ranged from 1.29 to 13.48 pgTEQ2005 g-1 lipid. The estimated geometric mean level for the sum of PCDD/F and dl-PCBs was 4.10 and 4.42 pgTEQ2005 g-1 lipid, lower-bound (LB) and UB respectively and were below the reference level reported by EFSA, which is associated with adverse effects in boys of 9 years and related to lactating mothers' exposure values of the tolerable weekly intake (TWI) of around 2 pgTEQ2005 kg-1 bw per week. Nevertheless, it was exceeded when the 95th percentile (8.31 pgTEQ2005 g-1 lipid, as UB) was considered. Results from a multiple regression analysis showed that age has a significant impact on milk ∑PCDD/Fs + dl-PCBs levels, with higher concentrations observed in the milk from older mothers.

Polybrominated diphenyl ethers in foods from the Region of Valencia: Dietary exposure and risk assessment.

Dietary exposure to polybrominated diphenyl ethers (PBDEs) of the population in the Region of Valencia, Spain, was assessed. A group of 320 composite samples of different fatty foods was collected and analyzed, including the following: vegetable oils, and foods of animal origin such as (a) fish and seafood, (b) eggs, (c) milk and dairy products, and (d) meat and meat products. Two scenarios were assumed for left-censored results: lower-bound (LB) and upper-bound (UB). Vegetable oils, and fish and seafood presented the highest content of PBDEs [mean values of 503 and 464 pg g-1 wet weight (ww) for total PBDEs, respectively, in the UB]. The dominating congeners were BDE47 in the food categories of fish and seafood, meat and meat products, and vegetable oils, and BDE99 in the categories of eggs, and milk and dairy products. The dietary exposure to PBDEs through consumption of the studied foods by the population in the Region of Valencia was estimated for adults (>15 years of age) and young people (6-15 years of age). Average intake levels (UB scenario) were 1.443 and 3.456 ng kg bw-1 day-1 for adults and young people, respectively. In a risk-assessment context, the margin of exposure (MOE) for congener BDE47, -99, -153, and -209 (ranged: 30-3E6) indicate that the current dietary exposure to these substances does not pose a risk to human health.

Biomonitoring of non-persistent pesticides in urine from lactating mothers: Exposure and risk assessment.

The aim of the present study was to assess the exposure to pesticides in urine from Spanish lactating mothers (n = 116). Six nonspecific (dialkyl phosphates) and 20 specific metabolites of organophosphate pesticides (OPs), herbicides and pyrethroids were analyzed. The most frequently detected biomarkers were diethyl phosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinol and 3-phenoxybenzoic acid, whose geometric means were 1.9 ng·mL-1, 0.8 ng·mL-1, 1.5 ng·mL-1 and 1.4 ng·mL-1, respectively. Herbicide metabolites were the least frequently detected biomarkers with detection frequencies between 0% (2,4,5-Trichlorophenoxyacetic acid) and 22% (2,4-Dichlorophenoxyacetic acid). Multiple regression analyses showed that the closeness to a farming activity, the place of residence and the presence of garden/plants at home were some of the most important contributors to urinary levels of pesticide metabolites. Estimated daily intake (EDI), hazard quotient (HQ) and hazard index (HI) were obtained in order to interpret urinary levels of the most frequently detected pesticide metabolites in a risk assessment context. The highest EDIs were obtained for chlorpyrifos (0.40-1.14 µg·kg bw-1·day-1) and deltamethrin (0.34-4.73 µg·kg bw-1·day-1). The calculated HQ for chlorpyrifos, dimethoate, parathion and deltamethrin ranged from 0.01 to 0.47, and HI for OPs ranged from 0.09 to 0.33 showing that apparently there were low health risks due to the exposure to these pesticides in this group of Spanish breastfeeding women.

Development of a novel methodology for determination of dialkyl phosphates in human urine using liquid chromatography-tandem mass spectrometry.

In this study a fast and sensitive method was developed for the quantitative determination of six dialkyl phosphates (DAPs) in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed methodology was based on liquid-liquid extraction (LLE) with diethyl ether and ethyl acetate using MgSO4 and NaCl, followed by liquid chromatography and mass spectrometry detection in the selected reaction mode (SRM) with negative electrospray ionization (ESI-). Detection settings were optimized by design of experiments (DoE). Dibutyl phosphate (DBP) was used as internal standard. Several criteria established in the SANTE/11813/2017 guidance document for pesticide residues and analysis in food and feed were used to validate the suggested method: recoveries (R %) between 70 and 120% and coefficients of variation (CV %) ≤ 20%. The procedural limit of quantification (LoQ) was 0.50 ng mL-1 for the six DAPs. The R% and CV% ranges were 82-117% and 3-20%, respectively. In total, 20 spot urine samples from lactating mothers were satisfactorily analyzed by the proposed analytical strategy.